4.6.4. Transformation, Selection and Verification

After PC8 extraction and EtOH precipitation, the ligation mixture was transformed into Escherichia coli (E. coli OneShot STLB-3 bacteria; Life Technologies) using standard electroporation procedure (Electroporator 2510; Eppendorf, Hamburg, Germany). Transformed cells were shaken for 1 h with 190 rpm at 37 °C in lysogeny broth (LB) medium (LB-broth, L2542; Sigma-Aldrich). The cells were plated on LB-agarose-agar (LB-broth; SELECT Agar, 30391049; Invitrogen) containing 100 µg/mL carbenicillin (C1389; Sigma) and incubated at 37 °C overnight. The next day, grown colonies were picked and incubated at 37 °C and 190 rpm for 4 h in 200 µL LB medium each in a 96-well plate and then centrifuged at 2500 rpm for 3 min (Multifuge 3 SR; Heraeus, Hanau, Germany). The pellet was resuspended in water and 1 µL of each bacteria suspension was added to a PCR plate well containing reaction mixture (1.5 µL 10× TrisA buffer, 0.6 µL MgCl₂ (50 mM), 0.3 µL dNTPs (10 mM), 0.5 µL primers (sense + antisense; 10 µM), 10.3 µL DEPC water and 0.3 µL Taq-polymerase (5000 units/µL per well)) for direct Bac PCR. The PCR was performed in a thermocycler first at 95 °C for 3 min following 35 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s. Primers used were DsRed-sense (AGC TGG ACA TCA CCT CCC ACA ACG) and pLJM-antisense (CCA ATG ACT TAC AAG GCA GC).

Following that, 5 µL of each PCR product were tested for positive clones using a TAE gel containing 1.5% agarose with 1 kb plus Ladder as a marker. Plasmid DNA minipreparations were made from overnight cultures using PureYield Plasmid Miniprep System (A1223; Promega), following the manufacturer’s protocol. The DNA concentration of the sample was measured photometrically and a test restriction of the mini prep was performed showing the expected fragments.

Finally, 10 µL of bacteria suspension were incubated in 150 mL LB medium and 150 µL carbenicillin overnight at 37 °C and 190 rpm and a midi-preparation was performed the subsequent day using PureYield Plasmid Midiprep System (A2492; Promega). The correct sequence was verified via standard sequencing and the resulting plasmid was stored at −20 °C until use. In the following, our product is called pLKO-miR204-DsRed.